Project Summary An alarming rise in opioids prescribed for pain relief during pregnancy has resulted in an increasing number of neonatal intensive care unit (NICU) admissions for neonatal drug withdrawal, also called neonatal abstinence syndrome (NAS). Evidence that these NAS babies are more likely to become ?addicted? later in life has emerged in recent years. Although the exact mechanism has not been identified, there is robust evidence that DNA methylation is altered in individuals taking chronic opioids, and polymorphisms and DNA methylation of OPRM1 promoter have recently been shown to influence NAS outcomes. Our hypothesis is that in utero opioid exposure results in epigenetic processes with potentially long term clinical consequences for the fetus/child such as an increased vulnerability to develop an ?opioid (mis)use disorder?. The rationale for the proposed pilot project is that evaluation of DNA methylation in neonates exposed in utero to opioids will lay the foundation to study the relationship between DNA methylation and other epigenetic processes with specific pain behaviors during childhood and drug addiction later in life. In this pilot exploratory longitudinal case-control study we will evaluate the impact of maternal opioid use for pain relief during pregnancy and consequent prolonged in utero opioid exposure in cases (20 mothers on prescribed opioids for pain relief and their neonate) and controls (20 unexposed mothers/babies). Maternal opioid exposure will be quantified using Liquid Chromatography? Tandem Mass Spectrometry at delivery (hair), and neonatal chronic exposure will be evaluated on meconium and hair samples at birth, and again at 2 months (hair). We propose 3 specific aims: (1) compare neonatal outcomes (NAS outcomes & pain during immunization in particular) between exposed vs unexposed babies, (2) assess neonatal DNA methylation patterns (OPRM1 & COMT promoters, LINE-1) at birth and 2 months and (3) evaluate opioid metabolism and genetic predictors (CYP2D6/3A4, OPRM1 & COMT) of neonatal outcomes and NAS management. Such an approach is innovative as repeated DNA methylation assays in neonates evaluating behavioral and pain phenotypes after in utero opioid exposure have not been undertaken and may (a) provide key insight into the mechanisms/pathways underlying opioid tolerance and drug addition, (b) help propose strategies to improve our ability to diagnose and treat opioid addiction and (c) prevent opioid exposure in individuals identified as being at risk for opioid dependence, tolerance and addiction due to genetic/epigenetic markers. Public Health Statement The proposed research is extremely timely because death from opioids prescribed for pain relief has become an epidemic in the U.S. causing 50 deaths every day and FDA?s recent safety announcement regarding risks associated with pain medicines use during pregnancy emphasizes the current ?over- exposure? to opioids in the U.S. Elucidating the long-term effects of in utero opioid exposure is key to further our understanding of the genetic and epigenetic contributions to opioid addiction, and potentially predict and prevent opioid misuse and chronic pain.